Novel insights into tumorigenesis revealed by molecular analysis of Lynch syndrome cases with multiple colorectal tumors

Alisa OlkinuoraSatu Mäki-NevalaSanjeevi UkwattageAri RistimäkiMaarit AhtiainenJukka-Pekka MecklinPäivi Peltomäki

Background: Lynch syndrome (LS) is an autosomal dominant multi-organ cancer syndrome with a high lifetime risk of cancer. The number of cumulative colorectal adenomas in LS does not generally exceed ten, and removal of adenomas via routine screening minimizes the cancer burden. However, abnormal phenotypes may mislead initial diagnosis and subsequently cause suboptimal treatment.

Aim: Currently, there is no standard guide for the care of multiple colorectal adenomas in LS individuals. We aimed to shed insight into the molecular features and reasons for multiplicity of adenomas in LS patients.

Methods: We applied whole exome sequencing on nine adenomas (ten samples) and three assumed primary carcinomas (five samples) of an LS patient developing the tumors during a 21-year follow-up period. We compared the findings to the tumor profiles of two additional LS cases ascertained through colorectal tumor multiplicity, as well as to ten adenomas and 15 carcinomas from 23 unrelated LS patients with no elevated adenoma burden from the same population. As LS associated cancers can arise via several molecular pathways, we also profiled the tumors for CpG Island Methylator Phenotype (CIMP), and LINE-1 methylation.

Results: All tumors were microsatellite unstable (MSI), and MSI was present in several samples derived from normal mucosa as well. Interestingly, frequent frameshift variants in RNF43 were shared among substantial number of the tumors of our primary case and the tumors of LS cases with multiple tumors but almost absent in our control LS cases. The RNF43 variants were completely absent in the normal tissue, indicating tumor-associated mutational hotspots. The RNF43 status correlated with the mutational signature SBS96. Contrary to LS tumors from the reference set with no elevated colorectal tumor burden, the somatic variants occurred significantly more frequently at C>T in the CpG context, irrespective of CIMP or LINE-1 status, potentially indicating other, yet unknown methylation-related mechanisms. There were no signs of somatic mosaicism affecting the MMR genes. Somatic variants in APC and CTNNB1 were unique to each tumor.

Conclusion: Frequent somatic RNF43 hot spot variants combined with SBS96 signature and increased tendency to DNA methylation may contribute to tumor multiplicity in LS.

PMID: 38725616

Mitotic abnormalities precede microsatellite instability in lynch syndrome-associated colorectal tumourigenesis

Marjaana Pussila, Aleksi Laiho, Petri Törönen, Pauliina Björkbacka, Sonja Nykänen, Kirsi Pylvänäinen, Liisa Holm, Jukka-Pekka Mecklin, Laura Renkonen-Sinisalo, Taru Lehtonen, Anna Lepistö, Jere Linden, Satu Mäki-Nevala, Päivi Peltomäki, Minna Nyström

Lynch syndrome (LS) is one of the most common hereditary cancer syndromes worldwide. Dominantly inherited mutation in one of four DNA mismatch repair genes combined with somatic events leads to mismatch repair deficiency and microsatellite instability (MSI) in tumours. Due to a high lifetime risk of cancer, regular surveillance plays a key role in cancer prevention; yet the observation of frequent interval cancers points to insufficient cancer prevention by colonoscopy-based methods alone. This study aimed to identify precancerous functional changes in colonic mucosa that could facilitate the monitoring and prevention of cancer development in LS.

The study material comprised colon biopsy specimens (n = 71) collected during colonoscopy examinations from LS carriers (tumour-free, or diagnosed with adenoma, or diagnosed with carcinoma) and a control group, which included sporadic cases without LS or neoplasia. The majority (80%) of LS carriers had an inherited genetic MLH1 mutation. The remaining 20% included MSH2 mutation carriers (13%) and MSH6 mutation carriers (7%). The transcriptomes were first analysed with RNA-sequencing and followed up with Gorilla Ontology analysis and Reactome Knowledgebase and Ingenuity Pathway Analyses to detect functional changes that might be associated with the initiation of the neoplastic process in LS individuals.

With pathway and gene ontology analyses combined with measurement of mitotic perimeters from colonic mucosa and tumours, we found an increased tendency to chromosomal instability (CIN), already present in macroscopically normal LS mucosa. Our results suggest that CIN is an earlier aberration than MSI and may be the initial cancer driving aberration, whereas MSI accelerates tumour formation. Furthermore, our results suggest that MLH1 deficiency plays a significant role in the development of CIN.

The results validate our previous findings from mice and highlight early mitotic abnormalities as an important contributor and precancerous marker of colorectal tumourigenesis in LS.